JP3151080B2 - Homogeneous enzyme immunoassay method, enzyme-labeled antibody and dry immunoassay element - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method for measuring trace components using an antigen-antibody reaction. More specifically, the present invention relates to a homogeneous enzyme immunization method for measuring an antigen (ligand) in a sample using an enzyme-labeled antibody, an enzyme-labeled antibody used in this analysis method, and a dry immunoassay element to which this analysis method is applied.

[0002]

BACKGROUND OF THE INVENTION The analysis of biological components, drugs and the like contained in body fluids such as blood and urine is very useful for diagnosing disease states and judging the course of treatment, and plays an important role in the field of clinical tests. As a method for analyzing such a trace component (ligand), an enzyme immunoassay (enzyme immunoassay)
There is. Enzyme immunoassays include a heterogeneous system that requires B / F separation and a homogeneous system that does not require B / F separation.

[0003] The heterogeneous system separates the antigen-antibody conjugate (B) generated by the antigen-antibody reaction and the unbound free antibody (and antigen) (F) by some method, and then separates the antigen-antibody conjugate from the antigen-antibody conjugate. Is to measure the labeling enzyme activity. According to this method, high sensitivity can be expected in principle because analysis is performed by separating the bound (B) and free form (F). However, the B / F separation is required, so that the analysis operation is complicated and the measurement time is long. Problem.

[0004] On the other hand, the homogeneous reaction is based on the fact that the enzyme activity of a labeling enzyme undergoes some interference when an antibody and an antigen (ligand) bind, and utilizes the enzyme inhibitory action by antigen-antibody binding. Generally, an enzyme is labeled with an enzyme, and a large molecule antibody binds to the antigen, thereby causing steric hindrance of the enzyme to bind to the substrate or enzymatic activity caused by a change in the three-dimensional structure of the enzyme. (For example, EMIT (Enzyme Multiplied Immun
oassay Technique)). When the antigen is a macromolecule, on the contrary, even if the antibody is labeled with an enzyme, suppression of the enzyme activity due to the antigen-antibody binding reaction can be detected. As described above, since the B / F separation is unnecessary in the uniform method, the operation is simple. However, there is a disadvantage that the sensitivity is lower than that of a non-uniform system in principle.

As a method for increasing the sensitivity of a homogeneous immunoassay, there is a method described in Japanese Patent Application Laid-Open No. 60-108756. In this method, a water-insoluble polymer substrate is used as an enzyme substrate.Because the enzyme reaction is performed on the substrate particle surface, that is, at the solid-liquid interface, the antibody labeled with the enzyme binds to the antigen. The effect of steric hindrance on the enzyme activity appears to be significant. However, even with this method, high-sensitivity detection is limited, and a more sensitive immunoassay method has been desired.

[0006] On the other hand, in clinical tests in which a large number of liquid samples are handled and routineized, it is desired that analysis can be performed easily and quickly and automatic operation can be performed. 49-53888, 59-7
7356, 59-102388, and U.S. Patent 4,459,358). Therefore, it is desirable that the analysis method be applicable to such a dry analysis element.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and it is a first object of the present invention to provide a homogeneous enzyme immunoassay method capable of performing a highly sensitive analysis by a simple operation. It is a second object of the present invention to provide an enzyme-labeled antibody used in this analysis method. A third object of the present invention is to provide a dry enzyme immunoassay element which can be analyzed quickly by applying this analysis method.

[0008]

SUMMARY OF THE INVENTION The first object of the present invention is to provide a homogeneous enzyme immunoassay method for measuring the amount of an antigen by measuring a change in enzyme activity caused by a reaction between the antigen and an enzyme-labeled antibody. Wherein a homogeneous enzyme immunoassay method comprising using an enzyme-labeled antibody having two or more monoclonal antibodies recognizing different epitopes of an antigen and an enzyme.

[0009] A second object of the present invention is to provide an enzyme-labeled antibody for homogeneous enzyme immunoassay, comprising a conjugate of two or more monoclonal antibodies recognizing different epitopes of an antigen and an enzyme. Can be achieved.

A third object of the present invention is to provide a dry immunoassay element for measuring an amount of an antigen by measuring a change in an enzyme activity caused by a reaction between the antigen and an enzyme-labeled antibody. And a dry immunoassay element comprising an immunoreactive layer having an enzyme-labeled antibody having two or more monoclonal antibodies recognizing the above and an enzyme.

[0011]

According to the present invention, instead of an enzyme-labeled antibody in which one kind of monoclonal antibody is bound to an enzyme molecule as in the prior art, a different enzyme that recognizes a different epitope (antigenic determinant) of a test antigen on one enzyme molecule is used. This is based on the findings of the inventor of the present invention who has found that the above-mentioned monoclonal antibody is bound and used as an enzyme-labeled antibody, so that homogeneous enzyme immunoassay can be performed with extremely high sensitivity. How the use of two or more types of monoclonal antibodies contributes to the improvement of sensitivity is not known in detail, but is probably due to the following reasons.

In general, when an enzyme-labeled antibody reacts with a test antigen, the enzyme active site of the labeled enzyme is sterically hindered by the antigen, and the enzyme activity is suppressed. In the enzyme-labeled antibody of the present invention, two or more different antibodies recognizing different epitopes (antigenic determinants) of the test antigen are bound to one enzyme molecule, and this is used as the enzyme-labeled antibody. Thus, in the present invention, in addition to just making an antigen-antibody conjugate,
Apparently multivalent antigen and heterogeneous multivalent antibody (2
(To which the above antibodies are bound), thereby forming a matrix structure formed through the antigen molecule and the enzyme molecule. Such a matrix structure
Further, the enzyme active site is covered, and it is considered that steric hindrance to the enzyme activity due to the antigen-antibody binding reaction appears strongly.

[0013] In a preferred embodiment, fragments such as Fab ', F (ab') ₂ or Fab are used as monoclonal antibodies. These fragments are preferred in that they have no Fc portion that causes noise. In particular, Fab 'fragments are particularly preferable because they have an SH group in the hinge region of the antibody and thus can be easily bound to an enzyme.

When the analysis method of the present invention is applied to a dry analysis element, the enzyme-labeled antibody can be contained in the same immunoreactive layer together with the substrate. In a dry analytical element, an enzymatic reaction does not occur in a dry state other than the time of use, so that it can be contained in the same layer. The enzyme activity of the labeled enzyme is inhibited by the matrix structure of the antigen and the enzyme-labeled antibody generated by the antigen-antibody reaction. Alternatively, the immunoreactive layer may contain only the enzyme-labeled antibody and another substrate layer containing the enzyme substrate may be provided. In this case, the matrix structure generated by the antigen-antibody reaction in the immune reaction layer is captured by the layer structure, and cannot be transferred to the next substrate layer. In any case, the amount of antigen in the sample can be detected as suppression of the enzyme reaction.

[0015]

Detailed Description of the Invention The object to be measured according to the present invention is a ligand having an antigenic determinant contained in a sample, that is, an antigen. The type of the specimen is not limited, for example, blood (whole blood, plasma, serum), lymph,
There is urine. The ligand may be an antigen having at least two or more different epitopes (antigenic determinants). However, the structure of these epitopes need not be clarified, and may be any as long as two or more monoclonal antibodies that can be distinguished from each other can be prepared. For example, plasma proteins such as C-reactive protein (CRP), albumin, immunoglobulin, and ferritin; viruses such as HB antigen; bacteria; various organs such as α-fetoprotein, and antigens present in blood and urine. .

Antibodies As antibodies, two or more monoclonal antibodies that recognize different epitopes of the test antigen are used.

The monoclonal antibody can be obtained by a usual method. That is, the antigen is injected several times into the abdominal cavity or the like together with the adjuvant, and the spleen cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. Then, an antibody-producing cell is cloned from the fused cells and grown as a monoclonal cell. By further injecting the proliferating cells intraperitoneally into the mouse, ascites and serum containing the monoclonal antibody can be obtained. The antibody can be easily purified by a combination of ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography (such as protein A-agarose), and gel filtration. The antibody subclass is
Examples include IgG1, IgG2a, IgG2b, etc.
IgG with high antibody fragmentation efficiency when preparing antibody conjugates
1 is particularly excellent. The obtained IgG1 was obtained by removing Fc site with a protease such as activated papain or pepsin.
As (ab ') ₂ , it can be further reduced to induce a Fab' fragment.

In a preferred embodiment, Fab 'fragments are used as monoclonal antibodies. An intact antibody (IgG) has Fab (antigen binding site) and Fc (complement binding site). When using an enzyme-labeled antibody by combining an intact antibody with an enzyme, if the sample is a blood sample,
Complement components in the blood bind to the Fc portion, causing steric hindrance and inhibiting enzyme activity. Even when the sample is not a blood sample, the Fc portion non-specifically adsorbs on the surface of pores and internal voids of a porous member constituting a vessel wall of the reaction vessel, an immune reaction layer, etc. The activity becomes apparently low and causes noise during measurement. In order to remove these noises, it is necessary to use Fa without the Fc portion.
It is preferred to use b ', F (ab') ₂ or Fab fragments as antibodies. Among them, it is most preferable to use a Fab ′ fragment having a free SH group with the antibody for convenience of binding to the enzyme.

Selection of monoclonal antibodies having different recognition epitopes is performed by ELISA (Enzyme-linked Immunosorben).
t assay) or the like. For example, an antigen is bound to a microtiter plate coated and sensitized with the monoclonal antibody 1, and a separately prepared biotinylated monoclonal antibody 2 is reacted. This is called avidin-POD (P
eroxidase). If the reaction is positive, it can be determined that antibody 1 and antibody 2 have different recognition epitopes.

The enzyme constituting the labeled enzyme / antibody complex can be selected in consideration of the combination with the enzyme substrate used in the subsequent enzymatic reaction. In the present invention, the reactivity with the enzyme reacting with the enzyme substrate is suppressed by steric hindrance due to the formation of a matrix-like structure of the enzyme / antibody / antigen. It is preferable to select a system in which is easily detected. That is, a relatively high molecular weight enzyme substrate is preferable in terms of sensitivity. For example, a substrate having a molecular weight of about 20,000 or more, preferably about 100,000 or more is used. Such substrates include starch as the substrate for the enzyme amylase; substrate cellulose for the enzyme cellulase; gelatin for the protease;
Proteins such as hemocyanin; and various fats and oils for lipase. Reports on the above-mentioned enzyme and substrate selections are disclosed in JP-A-60-108756, 60-171461, 60-171460.
In detail. Among them, amylase using starch as a substrate is preferable. In addition, when these substrates are water-insoluble substrates, steric hindrance due to the matrix-like structure of the enzyme / antibody / antigen appears remarkably, and it is particularly preferable to use these.

As the amylase, α-amylase, β-amylase
Amylase, glucoamylase, etc., which are not substantially contained in the sample are preferred from the viewpoint of noise prevention. Amylases originate in a wide range of animals (saliva, pancreatic juice, etc.), plants and microorganisms. Therefore, when analyzing body fluids, blood, etc. of humans and animals, it is preferable not to use amylase derived from higher animals.

Amylases derived from microorganisms and plants include glucoamylase derived from Aspergillus, Rhizopus, Saccharomyces, and the like; β-amylase derived from barley malt, wheat, soybean, and the like; Bacillus Subtilis), Streptomyces griseus, Pseudomonas stutzeri, Thermoactiomyces vulg
aris) and the like. Among them, Bacillus subtilis (Bac
α-amylase from illus Subtilis) is most preferred.

It is preferable that these enzymes are not affected by an interfering factor present in any of the samples, and it is preferable that there are no competing enzymes of the same type in the samples. However,
When an enzyme of the same kind as the labeling enzyme is contained in the sample, this enzyme inhibitor may be used. The enzyme inhibitor may be any as long as the degree of inhibiting the enzyme in the sample is greater than the degree of inhibiting the activity of the labeled enzyme. The enzyme inhibitor completely inactivates the enzyme in the sample, but most preferably does not inhibit the labeled enzyme at all. However, in practice, it is sufficient that the blank value is not simply increased at the time of measurement, and after the measurement, the enzyme activity in the sample may be recovered by deactivating the enzyme inhibitor. The enzyme inhibitor may be any as long as it does not inhibit the enzyme of the enzyme-labeled antibody, and may inhibit the enzyme in the free state. This enzyme inhibitor may be selected from known enzyme inhibitors having the above specificity. Alternatively, an antibody against a problematic enzyme in a sample may be prepared and used as an enzyme inhibitor.

When α-amylase is used as oxygen, carboxymethylated starch, starch, amylose, amylopectin and the like can be used as substrates. In particular, when a water-insoluble starch or the like is used, the enzymatic reaction is a reaction at the surface of the substrate particles, that is, at the solid-liquid interface. It is preferred in terms of. Alternatively, water-insoluble dye starch may be used to detect dye (dye) attached to soluble amylose which is a product of enzymatic decomposition. As such a water-insoluble blue starch polymer, commercially available products such as neoamylase (manufactured by Daiichi Kagaku) can be used.

Coupling of Enzyme and Antibody The binding method of the enzyme and the antibody can be carried out using functional groups (amino group, carboxyl group, thiol group, etc.) of two substances. Representative coupling methods include the glutaraldehyde method, the periodic acid method, the pyridyl disulfide method,
Maleimide-succinimide method and the like. The binding method is not limited to these examples, and in addition, for example, `` Method in Immunology and Immunochemistry '' Vol. 1,
(CAWilliams, MWChase, Academic Press, 1967)
Alternatively, it can be used by appropriately selecting from the methods described in books such as "Enzyme Immunoassay" edited by Ishikawa, Kawai and Miyai (Medical Publishing, 1978). Among these coupling methods, the maleimide-succinimide method of cross-linking the thiol group of the antibody hinge portion and the amino group of the enzyme is excellent in that the reaction efficiency is high and the antibody activity can be maintained.

In the maleimide-succinimide method, for example, an enzyme is bound to two kinds of Fab 'as follows. First, the amino group of the enzyme is maleimidated with a maleimide-succinimide reagent. After purifying this by gel filtration,
It is subjected to conjugation with an antibody having a thiol group (Fab '). At this time, two kinds of antibodies having different epitopes (Fab '
) Are together subjected to a coupling reaction. This conjugation reaction is preferably carried out in the range of 3 to 7 mol of the antibody to 1 mol of the enzyme. When Fab '(molecular weight of about 50,000) is used as an antibody and α-amylase (molecular weight of about 50,000) is used as an enzyme, the weight of α-amylase is 1/3 to 1 / l of the total Fab' amount.
It is preferable to carry out the binding reaction in the range of 7. This binding reaction usually proceeds at 4 ° C. to room temperature.

The produced enzyme-antibody complex (enzyme-labeled antibody) is purified by gel filtration and, if necessary, dried by a lyophilization method or the like. The binding ratio between the enzyme and each antibody is not limited to 1: 1 and may be any ratio depending on the purpose. Since an ordinary enzyme has many amino groups, a plurality of maleimide groups are introduced, and a plurality of antibody molecules are introduced into one enzyme molecule. Since it is necessary that at least one molecule of an antibody having a different epitope is bound to one molecule of the enzyme, the molar ratio of the antibody to the enzyme in the complex must be 2 or more. The molar ratio is preferably in the range of 4 to 5. When Fab '(molecular weight of about 50,000) is used as an antibody and α-amylase (molecular weight of about 50,000) is used as an enzyme, the complex has a molecular weight of 150,000 daltons or more, preferably 250,000 to 300,000 daltons. Is preferred in that the detection sensitivity is high.

Analysis Method (Wet Method) First, a conjugate of an antigen and an enzyme-antibody complex contained in a sample is brought into contact in a solution. At that time, the temperature of the solution is 20-45
It is generally appropriate that the temperature is in the range of about 4.0 to about 8.5. If necessary to keep the pH constant, a buffer such as a phosphate buffer or an acetate buffer may be used. The contact time between the antigen and the enzyme-antibody complex only needs to be sufficient to allow sufficient reaction. For example, when the temperature of the solution is 37 ° C, 20 to 30 minutes is appropriate. Thereafter, an enzyme substrate is added, and the enzyme activity of the enzyme-antibody complex is measured. If the test antigen is present in the sample, it can be detected as suppression of the enzyme activity. If a calibration curve is drawn in advance using a solution containing a known amount of the test antigen, the amount of the test antigen in the sample can be quantified.

Incidentally, only the reaction between the antigen and the enzyme-antibody complex may be carried out in a solution system, and the reaction solution after the reaction may be subjected to dry analysis. That is, the enzyme activity may be measured by preparing a dry analytical element having a substrate layer containing the enzyme substrate of the labeling enzyme, and spotting the reaction solution after the immunoreaction on the dry analytical element. As the layer configuration, a layer obtained by removing an immunoreactive layer of a dry analysis element described below can be used.

[0030] describing the applied dry immunoassay element and the analytical method of the layer structure then the invention of immunoassay element. FIG. 1 shows one embodiment of the immunoassay element of the present invention. In FIG. 1, reference numeral 10 denotes a light-transmitting support, on which a detection layer (or reagent layer) 12 and an immune reaction layer 14 are laminated. The immunoreactive layer 14 is formed of a water-permeable layer, and contains the enzyme-labeled antibody of the present invention and a non-diffusible substrate that is a substrate for the labeled enzyme. The reagent layer 12 is composed of a water-permeable layer and contains a reagent composition for detecting an enzyme reaction product (diffusible substance) diffused and transferred from the immune reaction layer 14. When the enzyme reaction product is a substance that can be directly detected, such as a colored substance, the layer 12 does not need to contain a detection reagent composition, and in this case, the layer 12 functions as a detection layer.

The sample (antigen) in the liquid sample supplied with the element spotted thereon reacts with the enzyme-labeled antibody in the immunoreactive layer 14 to form a matrix structure. Therefore, the enzymatic activity on the substrate contained in the same reactive immune layer 14 is suppressed. As a result, the amount of the antigen in the sample can be known from the amount of the enzyme reaction product detected in the reagent layer (or the detection layer) 12.

The enzyme-labeled antibody and the enzyme substrate may be contained in separate layers. In this case, as shown in FIG. 2, a water-permeable substrate layer 16 containing an enzyme substrate is disposed on a reagent layer (or detection layer) 12, and an immunoreaction containing an enzyme-labeled antibody is further provided thereon. The layer 14A is provided. In this case, the specimen (antigen) in the liquid sample spotted and supplied to the element reacts with the enzyme-labeled antibody in the immunoreactive layer 14A to form an antigen-antibody bond, and becomes substantially immobile. Enzyme-labeled antibody that did not bind to the antigen (or a matrix structure with a structure small enough not to be captured by the layer structure)
Moves to the next substrate layer 16. In any of the embodiments shown in FIGS. 1 and 2, the enzyme immunoreaction can be advanced in the element only by dropping the liquid sample on the element.

The above is the basic configuration of the dry immunoassay element of the present invention. For other layers that can be incorporated,
The layer structure can be the same as that of various known dry analysis elements. That is, the element includes an immunoreactive layer (or an immunoreactive layer and a substrate layer), a reagent layer (or a detection layer), a support, a spreading layer, a detection layer, a light shielding layer, an adhesive layer, a water absorbing layer, an undercoat layer, and the like. It may be a multilayer including layers. As such an analysis element, for example, JP-A-49-53888 (corresponding US Pat.
92,158), JP-A-51-40191 (corresponding U.S. Patent 4,042,33
5), and JP-A-55-164356 (corresponding U.S. Pat.
2) and JP-A-61-4959 (corresponding EPC published patent 0166365A).

When a light-permeable, water-impermeable support is used, the dry immunoassay element of the present invention can practically have the following configuration. However, the content of the present invention is not limited to this. (1) One having a reagent layer on a support and an immunoreactive layer thereon. (2) One having a reagent layer, a substrate layer, and an immunoreactive layer on a support in this order. (3) One having a reagent layer, an adhesive layer, and an immunoreactive layer on a support in this order. (4) One having a reagent layer, an adhesive layer, a substrate layer, and an immunoreactive layer on a support in this order. (5) One having a detection layer, a reagent layer, and an immunoreactive layer on a support in this order. (6) One having a detection layer, a reagent layer, a substrate layer, and an immunoreactive layer on a support in this order. (7) One having a reagent layer, a light reflection layer, and an immunoreactive layer on a support in this order. (8) One having a reagent layer, a light reflection layer, a substrate layer, and an immunoreactive layer on a support in this order. (9) One having a detection layer, a reagent layer, a light reflection layer, and an immunoreactive layer on a support in this order. (10) A support having a detection layer, a reagent layer, a light reflection layer, a substrate layer, and an immunoreactive layer in this order on a support. (11) One having a detection layer, a light reflection layer, a reagent layer, and an immunoreactive layer on a support in this order. (12) One having a detection layer, a light reflection layer, a reagent layer, a substrate layer, and an immunoreactive layer in this order on a support. (13) One having a second reagent layer, a light reflecting layer, a first reagent layer, and an immunoreactive layer on a support in this order. (14) One having a second reagent layer, a light reflecting layer, a first reagent layer, a substrate layer, and an immunoreactive layer on a support in this order. (15) One having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, and an immunoreactive layer on a support in this order. (16) One having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, a substrate layer, and an immunoreactive layer in this order on a support.

In the above (1) to (12), the reagent layer may be composed of a plurality of different layers. A water absorbing layer may be provided between the support and the reagent layer or the detection layer. Further, a filtration layer may be provided between each layer. Further, a developing layer may be provided on the substrate layer, or the substrate layer may have a developing action and function as a developing layer. When a solid component such as blood cells is present in the sample, a suitable filtration layer may be provided on the uppermost layer of the analysis element.

The immunoreactive layer and the substrate layer The immunoreactive layers 14, 14A and the substrate layer 16 are composed of a water-permeable layer. In order to ensure the water permeability of these layers, it is preferable to use a porous layer made of a porous medium or a layer made of a hydrophilic polymer binder.

The porous layer may be fibrous or non-fibrous. Examples of the fibrous material include filter paper, nonwoven fabric, woven fabric (for example, plain woven fabric), knitted fabric,
(Eg, tricot knitted fabric), glass fiber filter paper, and the like. As a non-fibrous material, JP-A-49-5
3888 etc., membrane filters made of cellulose acetate and the like, JP-A-49-53888, JP-A-55-90859 (corresponding US Pat. No. 4,258,001), JP-A-58-70163 (corresponding US Pat.
86, 537), etc., and may be any of continuous void-containing granular structure layers composed of inorganic or organic fine particles. JP 61
-4959 (corresponding European published EP 0166365A), JP-A-62-116258
JP-A-62-138756 (corresponding European publication EP 0226465A), JP-A-62-138757 (corresponding European publication EP 0226465A),
Also suitable are laminates of a plurality of partially bonded porous layers as described, for example, in EP-A-138758 (corresponding European publication EP 0226465A).

The porous layer may be a spreading layer having a so-called metering action in which the liquid spreads in an area approximately proportional to the amount of the supplied liquid. The spreading layer is preferably a woven fabric, a knitted fabric, or the like. A woven fabric or the like may be subjected to a glow discharge treatment as described in JP-A-57-66359. In order to adjust the development area, the development speed, etc., in the development layer, JP-A-60-222770 (corresponding: EP 0162301A), JP-A-63-219397 (corresponding to West German Patent Publication DE 37 17 913A), -112999 (correspondence: DE 37 17 913A), JP-A-62-1
82652 (corresponding to DE 37 17 913A) may contain a hydrophilic polymer or a surfactant.

As a useful method of incorporating the substrate into the porous layer, for example, paper, cloth, a porous membrane made of a polymer, or the like is preliminarily impregnated or coated with the substrate, and then the other water permeable layer provided on the support is used. It is also a useful method to adhere to a layer such as a reagent layer by a method as described in JP-A-55-164356. Alternatively, the porous layer may be replaced with another water-permeable layer (eg, a reagent layer).
After adhering to the porous layer by the method described above, a composition containing a substrate may be applied to the porous layer. Known methods can be used for impregnation or application to the porous layer. For the coating, for example, dip coating, doctor coating, hopper coating, curtain coating or the like is appropriately selected and used. The thickness of the substrate layer thus produced is not particularly limited, but when provided as a coating layer,
A range of about 1 μm to 50 μm, preferably 2 μm to 30 μm is appropriate. When using a method other than coating, such as lamination by lamination, the thickness can vary greatly in the range of several tens of μm to several hundreds of μm.

When the immunoreactive layers 14, 14A and the substrate layer 16 are composed of a water-permeable layer made of a hydrophilic polymer binder, the following hydrophilic polymers can be used, for example. Gelatin and derivatives thereof (eg, phthalated gelatin), cellulose derivatives (eg, hydroxyethyl cellulose), agarose, sodium alginate, acrylamide copolymers, methacrylamide copolymers, copolymers of acrylamide or methacrylamide with various vinylic monomers And polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, and copolymers of acrylic acid and various vinylic monomers.

A substrate layer composed of a hydrophilic polymer binder is disclosed in JP-B-53-21677 (corresponding to US Pat. No. 3,992,158),
JP-A-55-164356 (corresponding U.S. Patent 4,292,272);
54-101398 (corresponding U.S. Patent 4,132,528), JP-A-61-292
No. 063 (Chemical Abstracts, 106 , 210567y), etc., an aqueous solution or aqueous dispersion containing a substrate or other reagent composition and a hydrophilic polymer is coated on a support or another layer such as a detection layer. And dried. The dry thickness of the substrate layer using a hydrophilic polymer as a binder is about 2 μm to about 50 μm, preferably about 4 μm.
m ~ about 30μm range, the coating amount of about ² g / m 2 ~ about 50 g / m ^2,
Preferably from about 4 g / m ² ~ about 30 g / m ^2.

In the substrate layer, in addition to the non-diffusible substrate, an activator of an enzyme, a coenzyme, an interface, etc. are used for the purpose of improving various properties such as coating characteristics, diffusibility of a diffusible compound, reactivity, and storage stability. An activator, a pH buffer composition, a fine powder, an antioxidant, and other various additives composed of an organic or inorganic substance can be added. Examples of buffering agents that can be contained in the substrate layer are described in Chemical Chemistry Handbook, Basic Edition, edited by The Chemical Society of Japan (Tokyo, Maruzen Co., Ltd., 1966), pages 1312-1320, RMCDa.
wson et al ed., “Data for Biochemical Research”, 2nd edition (Oxford at the Clarendon Press, 1969) 47
6-508, `` Biochemistry '' 5 , 467-477 (1966), `` A
nalytical Biochemistry ", 104 , pp. 300-310 (1980). A buffer containing tris (hydroxymethyl) aminomethane (Tris) as a specific example of the pH buffer; a buffer containing phosphate; a buffer containing borate;
Good buffers such as a buffer containing citric acid or citrate; a buffer containing glycine; a buffer containing bicine; a buffer containing HEPES; a buffer containing MES, and the like.

The immunoreactive layer can be constituted in the same manner as the above-mentioned substrate layer. In order to allow the substrate and the enzyme-labeled antibody to be contained in one layer or two adjacent layers in a substantially dry state or substantially in the absence of water, the enzyme-labeled antibody may be contained in an alcohol (eg, ethanol) or the like. What is necessary is just to dissolve or disperse in a non-aqueous solvent and impregnate the water-permeable layer.

Reagent Layer and Detection Layer The reagent layer 12 contains a reagent composition for detecting a diffusible substance that has diffused and migrated from the immune reaction layer 14 (or the substrate layer 16). If necessary, the reagent composition contains a low-molecular-weight enzyme, and contains a detection reagent composition for detecting a low-molecular product generated by reducing the molecular weight of a diffusible substance. The reagent layer is composed of a water-permeable layer, and is preferably a continuous layer made of a hydrophilic polymer binder among the water-permeable layers described in the description of the substrate layer. The hydrophilic polymer binder to be used is determined in consideration of a diffusible product generated in the substrate layer, a coloring reagent contained in the reagent layer, and the like.

If the diffusible substance diffused and transferred from the immune reaction layer 14 (or the substrate layer 16) is directly detectable, the layer 12 does not need to contain the detection reagent composition. Functions as a detection layer. Even when the detection layer, among the water-permeable layers described in the description of the substrate layer,
It is preferable to form a continuous layer made of a hydrophilic polymer binder.

Support The support 10 may be any of light-impermeable (opaque), semi-transmissive (semi-transparent), and light-transmissive (transparent), but is generally light-transmissive. A water-impermeable support is preferred. Preferred materials for the light-transmitting water-impermeable support are polyethylene terephthalate and polystyrene. In order to firmly adhere the hydrophilic layer, an undercoat layer is usually provided or a hydrophilic treatment is performed.

Labeling enzyme-Non-diffusible substrate-Small molecule enzyme The dry immunoassay element of the present invention is disclosed in JP-A-3-295466 (corresponding to E
Similarly to P0451848A), 4-128655, and 4-765551, the reagent layer 12 contains a low-molecular-weight enzyme that further reduces the molecular weight of a diffusible substance decomposed by a labeling enzyme from a non-diffusible substrate. The sensitivity can be increased. In these combinations, the enzyme acts on the non-diffusible substrate to generate a diffusible substance, and the diffusible product can be easily detected by generating a lower molecular product by the below-described low molecular weight enzyme. You can choose from such combinations.

That is, an antibody-labeling enzyme is selected which decomposes a non-diffusible substrate composed of a high molecule and generates a diffusible product which further produces a low-molecular product by the low-molecular-weight enzyme. The non-diffusible substrate is selected from those that are non-diffusible (insoluble) in the aqueous sample liquid and do not themselves diffuse or migrate from the immune reaction layer 14 (or the substrate layer 16) to the reagent layer 12. Furthermore, the low-molecular-weight enzyme can be
The diffusible product generated from the non-diffusible substrate is selected from those that further reduce the detectable low molecular weight product. Hereinafter, these examples will be specifically described.

Enzyme As such an enzyme, a degrading enzyme that generates a diffusible oligomer from a non-diffusible substrate composed of a polymer is preferable, and among the above-mentioned labeling enzymes, for example, a carbohydrate hydrolase is preferable. . As such carbohydrate hydrolases,
α-amylase, β-amylase, glucoamylase,
There is lysozyme.

Non-diffusible substrate Examples of the substrate for the above-mentioned α-amylase, β-amylase and glucoamylase include carboxymethylated starch and starch. When carboxymethylated starch or starch is used as a non-diffusible substrate, a combination using α-amylase as a labeling enzyme and glucoamylase or α-glucosidase described later as a low-molecular-weight enzyme can be used.

Low-molecular-weight enzyme This low-molecular-weight enzyme may be the same kind of enzyme as the labeling enzyme. In this case, the labeling enzyme is an enzyme having an endo activity that cleaves from inside the molecule to generate an oligomer,
It is preferable that the low molecular weight enzyme has an exo activity that acts from the end of the molecule to form a monomer. For example, when the non-diffusible substrate is a polymer (for example, starch), one that can decompose a diffusible oligomer (for example, maltose) generated by a labeling enzyme into a monomer (for example, glucose) is used. Examples of such low-molecular-weight enzymes include sugar hydrolases, more specifically, α-amylase, β-amylase, glucoamylase, α-glucosidase and the like. When carboxymethylcellulose and cellulase are used as the non-diffusible substrate and the labeling enzyme, the C1 enzyme can be used as a low-molecular-weight enzyme.

Combinations of these labeling enzymes, non-diffusible substrates, and low molecular weight enzymes are described in known literature (eg, “Enzyme Handbook” (published by Bunji Maruo and Nobuo Tamiya, Asakura Shoten, 1982) and “Biochemical Handbook”. (Nobumasa Imura, other editions, Maruzen
1984)).

The low molecular weight product produced by the low molecular weight enzyme in the reagent layer can be optically detected by a known detection system reagent. As a method for detecting glucose finally produced by the low-molecular-weight enzyme, for example, a method for detecting hydrogen peroxide produced by oxidizing glucose in the presence of glucose oxidase (for example, Ann.
n. Biochem., 6 , 24 (1964), J. Clin. Pathol., 22 , 246 (19
69), Trinder reagent described in JP-A-49-50991 (corresponding U.S. Pat.No. 3,886,045), U.S. Pat.
No. 64356 (corresponding U.S. Patent 4,292,272)
Reagents, JP-A-53-26188 (corresponding U.S. Pat.No.4,089,747), reagents containing triaryl-substituted imidazole leuco dyes described in JP-A-58-45557, etc., JP-A-59-193352 (corresponding to European Patent Publication EP 0122641A), a method using a reagent containing a diaryl-monoaralkyl-substituted imidazole leuco dye described in JP-A-60-224677 (corresponding to U.S. Pat. No. 4,665,023), and detecting NADH formed in the presence of glucose dehydrogenase and NAD. A known method such as a method and a method of detecting glucose-6-phosphate generated in the presence of hexokinase can be used. Among these detection methods, a method of detecting hydrogen peroxide generated by oxidizing glucose in the presence of glucose oxidase using peroxidase and a leuco dye is most desirable in terms of sensitivity.

These detection reagents are applied to the reagent layer 12 of the analysis element.
May be contained together with a low-molecular-weight enzyme.
The layer may be contained in another layer provided below the second layer (for example, the second reagent layer or the detection layer), and detection may be performed by this layer.
When a leuco dye is used, a dispersion of a solution of a water-immiscible solvent in a hydrophilic binder is preferable from the viewpoint of the stability of the formed dye.

Method for Producing Immunoassay Element The dry immunoassay element of the present invention can be prepared by a known method described in the aforementioned patent specifications. The analysis element of the present invention is cut into small pieces such as a square or a circle of approximately the same size having a side of about 15 mm to about 30 mm, and Japanese Patent Publication No. 57-28331 (corresponding U.S. Pat.
4,387,990), Japanese Patent Laid-Open No. 57-63452, Japanese Utility Model Application No. 58-32350,
8-501144 (corresponding international publication: WO 83/00391) or the like is preferably used in a slide frame as a chemical analysis slide from the viewpoint of production, packaging, transport, storage, measurement operation and the like. Depending on the purpose of use, it may be used in the form of a long tape in a cassette or magazine, or may be used by sticking or storing a small piece on a card having an opening.

Analysis Method Using Immunoassay Element The analysis element of the present invention enables the quantitative analysis of a high molecular antigen, which is a test substance, in a liquid sample by the same operation as that described in the above-mentioned various patent specifications. For example, an aqueous liquid sample solution such as plasma, serum, urine or the like in the range of about 5 μL to about 30 μL, preferably 8 to 15 μL is spotted on the substrate layer 14. The spotted analytical element is placed at a constant temperature in the range of about 20C to about 45C, preferably about 30C.
Incubate at a constant temperature in the range of 内 40 ° C. for 1-10 minutes. The color development or discoloration in the element is reflected and measured from the side of the light-transmitting support, and the amount of the macromolecular antigen in the sample can be determined by the principle of colorimetry using a previously prepared calibration curve. The quantitative analysis can be performed with high precision by keeping the amount of the liquid sample to be spotted, the incubation time and the temperature constant. The measurement operation is described in JP-A-60-125543, 60-220862,
Highly accurate quantitative analysis can be performed by a very easy operation using the chemical analyzer described in JP-A-61-294367. Note that, depending on the purpose and required accuracy, the degree of color development may be visually determined to perform semi-quantitative measurement. When the enzyme-labeled antibody is not contained in the analysis element, the aqueous sample solution is mixed with the solution containing the enzyme-labeled antibody before spotting on the element, and the binding reaction is sufficiently performed. Just wear it.

[0057]

Synthesis Example 1 Preparation of GMB Amylase A maleimide group was introduced into α-amylase as follows. To 1 mL of a 10 mg / mL B. subtilis α-amylase solution (0.1 M glycerophosphate buffer, pH 7.0), add GMBS (N-
(γ-maleimidobutyryloxy) succinimide; Dojin Chemical
Was added, and the mixture was reacted at room temperature for 2 hours. This reaction solution was subjected to gel filtration through a column of Sephadex G-25, and a flow-through fraction was collected.
-Maleimidobutylyloxy) amidated amylase (GM
B-amylase) was obtained. The concentration of the resulting GMB-amylase solution was 1.12 mg / mL.

[0058]

[Synthesis Example 2] Preparation of anti-human CRP • IgGFab '(first antibody: Fab'-
α) Two types of monoclonal antibody IgG against human CRP
Are antibodies obtained by fusing immunocytes (spleen cells) obtained by immunizing a mouse with mouse myeloma cells and cloning, and obtained by a conventional method. Among the obtained monoclonal antibodies, the following Fab ′ fragments were prepared for two kinds of antibodies (first antibody and second antibody) confirmed to have different recognition epitopes.

First anti-human CRP antibody IgG, 4.4 mg
Was dissolved in 1 mL of 0.1 M acetate buffer (pH 5.5), and 132 μg of activated papain was added thereto, followed by stirring at 37 ° C. for 2 hours.
This reaction solution was previously added to a 0.1 M phosphate buffer (pH 6.0; 1 mM ED).
The solution was applied to a Superdex-200 gel column equilibrated with TA-2Na) and eluted with the same phosphate buffer. Molecular weight
The peak eluted at around 100,000 was collected and subjected to anti-human CRP.
-IgGF (ab ') ₂ was obtained. Obtained anti-human CRP • IgG
5 mL of 0.1 M phosphate buffer (pH 6.0) containing 2.2 mg of F (ab ') ₂
Then, 100 μL of a 113 mg / mL aqueous solution of 2-mercaptoethylamine hydrochloride was added thereto, followed by stirring at 37 ° C. for 2 hours. This reaction solution was previously added to a 0.1 M glycerophosphate buffer (pH 7.0; 5 mM EDTA).
The mixture was subjected to gel filtration with Sephadex G-25 equilibrated with -Ca (containing Ca), and a flow-through fraction was collected. Obtained anti-human CRP
The IgGFab '(hereinafter referred to as Fab'-α) fraction was diluted with the same buffer to a concentration of 0.1 mg / mL.

[0060]

[Synthesis Example 3] Preparation of anti-human CRP · IgG Fab _'2 (second antibody: Fab'
β) 5.3 mg of the second anti-human CRP antibody IgG was added to 1 mL of 0.1
Dissolve in M acetate buffer (pH 5.5) and add activated papain 1
59 μg was added and the mixture was stirred at 37 ° C. for 2 hours. This reaction solution is
The solution was applied to a Superdex-200 gel column previously equilibrated with a 0.1 M phosphate buffer (pH 6.0; containing 1 mM EDTA-2Na), and eluted with the same phosphate buffer. A peak portion eluted at a molecular weight of around 100,000 was collected and subjected to anti-human CRP.IgGF (ab ') ₂
I got Obtained anti-human CRP-IgGF (ab ') ₂ 3.1
113 mg / mL in 5 mL of 0.1 M phosphate buffer (pH 6.0) containing
100 μL of 2-mercaptoethylamine hydrochloride aqueous solution
The mixture was stirred at 37 ° C. for 2 hours. 0.1 M
The mixture was subjected to gel filtration with Sephadex G-25 equilibrated with a glycerophosphate buffer (pH 7.0; containing 5 mM EDTA-Ca), and a flow-through fraction was collected. The obtained anti-human CRP.IgGFab '(hereinafter referred to as Fab'-β) fraction was diluted with the same buffer to a concentration of 0.1%.
mg / mL.

[0061]

Synthesis Example 4 Preparation of α-Amylase / Fab′-α / Fab′-β Conjugate Two kinds of Fab ′ fragment (Fab′-α and Fab′-β) solutions prepared in Synthesis Examples 2 and 3 were prepared. Mix 3.65 mL, add 108 μL of GMB-amylase solution prepared in Synthesis Example 1
Was added and reacted at 4 ° C. for 20 hours. This reaction solution is
The solution was applied to Superdex-200 equilibrated with 0.1 M glycerophosphate (pH 7.0; containing 0.5 mM EDTA-Ca) and eluted with the same buffer. The fraction having a molecular weight of about 270,000 was fractionated to obtain a target enzyme-labeled antibody (α-amylase / Fab′-α / Fab′-β conjugate). The molecular weight of this conjugate corresponds to one in which four molecules of Fab'-α and Fab'-β are bonded to one molecule of the enzyme.

[0062]

Synthesis Example 5 Preparation of α-Amylase / Fab′-α Conjugate (Comparative Example 1) As a comparative example, an enzyme / antibody conjugate which was conjugated to only one type of monoclonal antibody was prepared. The GMB-modified amylase prepared in Synthesis Example 1 was added to 3.65 mL of a 0.1 mg / mL solution of anti-human CRP.IgGFab '(Fab'-α) obtained in Synthesis Example 2 in an amount of 54 μm.
L was added and reacted at 4 ° C. for 20 hours. This reaction solution was previously added to 0.1 M glycerophosphate (pH 7.0; containing 0.5 mM MEDTA-Ca).
The solution was applied to Superdex-200, which had been equilibrated with, and eluted with the same buffer. The fraction having a molecular weight of about 270,000 was fractionated to obtain an enzyme-labeled antibody (α-amylase / Fab′-α conjugate) of Comparative Example 1. The molecular weight of this conjugate corresponds to one in which four molecules of Fab′-α are bonded to one molecule of the enzyme.

[0063]

[Synthesis Example 6] Preparation of α-amylase / Fab'-β conjugate (Comparative Example 2) As a comparative example, an enzyme / antibody conjugate conjugated only with another type of monoclonal antibody was prepared. Synthesis Example 3
Of the anti-human CRP-IgGFab '(Fab'-β) obtained in
To 3.65 mL of the 1 mg / mL solution, 54 μL of the GMB-amylase prepared in Synthesis Example 1 was added and reacted at 4 ° C. for 20 hours. This reaction solution was previously added to 0.1 M glycerophosphate (pH 7.0; 0.5 mM EDTA).
-Ca-containing) and applied to Superdex-200 equilibrated with the same buffer. A fraction having a molecular weight of about 270,000 was fractionated and subjected to the enzyme-labeled antibody (α-amylase / Fa) of Comparative Example 2.
b′-β conjugate) was obtained. The molecular weight of this conjugate is
This corresponds to one in which four molecules of Fab'-β are bonded to a molecule.

[0064]

Example 1 Measurement of Human CRP (Wet Method) Using the obtained enzyme-labeled antibody, human CRP was subjected to homogeneous enzyme immunoassay. 3 in the concentration range of 0 to 3.7 μg / mL
The α-amylase / Fab′-α / Fab′-β conjugate prepared in Synthesis Example 4 was added to 50 μL of the human CRP solution diluted twice, and reacted at 37 ° C. for 20 minutes. On the other hand, one tablet (45 mg blue starch, containing 3 mg BSA) of Neo Amylase Test "Daiichi" (Daiichi Pure Chemicals) was dissolved in 4 mL of 50 mM maleic acid buffer (pH 6.5), and 1 mL of this solution was added. Was added to the above reaction solution, and the mixture was further reacted at 37 ° C. for 1 hour. 5 mL 0.5
The reaction was stopped by adding an aqueous solution of N NaOH, and after stirring this,
The mixture was centrifuged at 3,000 rpm for 5 minutes to obtain a supernatant. The amount of the blue dye solubilized in the supernatant by the enzyme reaction was measured by absorbance measurement at 620 nm. Comparative Example 1 obtained in Synthesis Examples 5 and 6,
Human CRP was measured in the same manner for the enzyme-labeled antibody No. 2.

FIG. 3 shows a calibration curve showing the relationship between the obtained absorbance and the concentration of human CRP. As shown in FIG. 3, the example (− ○ −) was compared with the comparative example 1 (− ■ −) and the comparative example 2 (− ■ −).
The calibration curve of CRP was shifted to a lower concentration side than □-). This proves that Examples in which two kinds of antibodies having different epitopes are bound to one molecule of the enzyme are more effective for highly sensitive detection than Comparative Examples 1 and 2 in which one kind of monoclonal antibody is bound. Was

[0066]

Example 2 Thickness 180 provided with gelatin undercoat layer
A reagent solution containing a crosslinking agent was applied onto a μm colorless and transparent polyethylene terephthalate (PET) sheet (support) so as to have the following coating amount, and dried to form a reagent layer. Alkali-treated gelatin 14.5 g / m ² Nonylphenoxypolyethoxyethanol (containing 9 to 10 oxyethylene units on average) 0.2 g / m ² Glucose oxidase 5000 u / m ² Peroxidase 15000 u / m ² Glucoamylase 5000 u / m ² 2- (4-hydroxy-3,5-dimethoxyphenyl-4- [4- (dimethylamino) phenyl] -5-phenethylimidazole (leuco dye) acetate 0.38 g / m ² bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1 g / m ²

On this reagent layer, an adhesive layer was applied so as to have the following coating amount and dried to form an adhesive layer. Alkali-treated gelatin 14.5 g / m ² Bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1 g / m ²

Then, the following reagent-containing aqueous solution was applied to the surface of the adhesive layer so as to have the following coating amount, the gelatin layer was swollen, and PET spun yarn equivalent to 50 denier was knitted with 36 gauge yarn and was about 250 μm thick. The tricot knitted fabric was substantially uniformly lightly laminated under light pressure to provide a porous spreading layer. Nonylphenoxypolyethoxyethanol (containing 9 to 10 oxyethylene units on average) 0.15 g / m ² bis [(vinylsulfonylmethyl carbonyl) amino] methane 0.4 g / m ²

Next, a substrate was applied so as to have the following coating amount and dried to provide a substrate layer, thereby preparing a multilayer analytical element for CRP analysis. Carboxymethylated starch 5 g / m ² Nonylphenoxypolyethoxyethanol (containing an average of 9 to 10 oxyethylene units) 0.2 g / m ² Next, this analytical element was cut into 15 mm square chips and described in JP-A-57-63452. Of the present embodiment, the multilayer dry slide 1 for CRP analysis of this example was obtained.

[0070]

[Performance evaluation test] α-amylase / Fab'-α of Synthesis Example 4
The / Fab'-β conjugate was added to a 50 mM glycerophosphate buffer solution (pH 7) containing a known amount of CRP at a concentration of 0.1 mg / mL, and incubated at 37 ° C for 20 minutes. Thereafter, 10 μL of this solution was spotted on the slide 1 and kept at 37 ° C.
The reflection optical density at 650 nm was measured from the ET support side. Difference in reflection optical density 3 minutes and 5 minutes after spotting (ΔOD
_5-3 ) was obtained and a calibration curve was created. The enzyme-labeled antibody (α-amylase / Fab′-α) of Comparative Example 1 obtained in Synthesis Examples 5 and 6
Conjugate), the enzyme-labeled antibody of Comparative Example 2 (α-amylase /
The same operation was performed for the Fab'-β conjugate to prepare a calibration curve. FIG. 4 shows the obtained calibration curves. As shown in FIG. 4, Example 2 (−） −) is Comparative Example 1 (− （−).
−) And Comparative Example 2 (− □ −), the calibration curve of CRP shifted to a lower concentration side, indicating that highly sensitive detection was possible.

[0071]

Example 3 A multilayer analytical element was prepared in exactly the same manner as in Example 2, and the tricot knitted fabric layer serving as the substrate layer and the spreading layer was further subjected to the enzyme-labeled antibody (α-amylase / Fab) synthesized in Synthesis Example 4. The '-α / Fab'-β conjugate) was coated with an ethanol solution so as to have a coating amount of 3 mg / m ² , impregnated, and dried to prepare a multilayer analysis slide 2 for CRP analysis.

As comparative slides, the α-amylase / Fab′-α conjugate prepared in Synthesis Example 5 or the α-amylase / Fab′-β conjugate prepared in Synthesis Example 6 was used as the enzyme-labeled antibody. Comparative slides 3 and 4 having exactly the same configuration as slide 2 were prepared.

[0073]

[Performance evaluation test] Each slide 2, 3, 4 has a known amount of CR
10 μL of a 50 mM glycerophosphate buffer solution (pH 7) containing P was spotted at 37 ° C., and a center wavelength of 65 μm was applied from the PET support side.
The reflection optical density was measured with 0 nm visible light, and the difference (ΔOD _6-4 ) between the reflection optical densities at 4 minutes and 6 minutes after spotting was determined to prepare a calibration curve. As shown in FIG. 5, the calibration curve of CRP is lower on slide 2 (-３-) of Example 3 than on slide 3 (-■-) and slide 4 (-□-) for comparison. Was shifting. This indicates that the dry analysis element can be made more sensitive by applying the analysis method of the present invention.

[0074]

As described above, the enzyme-labeled antibody of the present invention
It is obtained by combining two or more monoclonal antibodies recognizing different epitopes of an antigen with an enzyme. Therefore, when this is used as an enzyme-labeled antibody in a homogeneous enzyme immunoassay, analysis can be performed with extremely high sensitivity. In addition, since the method is a homogeneous analysis method that does not require B / F separation, the analysis operation is extremely simple. In addition, by applying this analysis method to a dry analysis element, highly sensitive analysis can be performed simply and quickly.

The preferred embodiments of the present invention are summarized as follows. (1) In a homogeneous enzyme immunoassay for measuring the amount of an antigen by measuring a change in enzyme activity caused by a reaction between an antigen and an enzyme-labeled antibody, two or more monoclonal antibodies recognizing different epitopes of the antigen A homogeneous enzyme immunoassay method comprising using an enzyme-labeled antibody having an enzyme and an enzyme. (2) The homogeneous enzyme immunoassay according to (1), wherein the monoclonal antibody is a Fab ′ fragment. (3) The homogeneous enzyme immunoassay according to (1), wherein the monoclonal antibody is an F (ab ') ₂ fragment. (4) The method for homogeneous enzyme immunoassay according to (1), wherein the monoclonal antibody is a Fab fragment. (5) The homogeneous enzyme immunoassay according to (1), wherein a polymer substrate having a molecular weight of about 20,000 or more is used as a substrate for the enzyme. (6) The homogeneous enzyme immunoassay according to (1), wherein a water-insoluble substrate is used as the enzyme substrate. (7) An enzyme-labeled antibody for homogeneous enzyme immunoassay, comprising a conjugate of two or more monoclonal antibodies recognizing different epitopes of an antigen and an enzyme. (8) The enzyme-labeled antibody for homogeneous enzyme immunoassay according to (7), wherein the monoclonal antibody is a Fab ′ fragment. (9) The enzyme-labeled antibody for homogeneous enzyme immunoassay according to (7), wherein the monoclonal antibody is an F (ab ') ₂ fragment. (10) The enzyme-labeled antibody for homogeneous enzyme immunoassay according to (7), wherein the monoclonal antibody is a Fab fragment. (11) In a dry immunoassay element for measuring an amount of an antigen by measuring a change in enzyme activity caused by a reaction between an antigen and an enzyme-labeled antibody, two or more monoclonal antibodies and enzymes recognizing different epitopes of the antigen are used. A dry immunoassay element comprising an immunoreactive layer containing an enzyme-labeled antibody having the following formula: (12) The dry immunoassay element according to (11), wherein the immunoreactive layer contains a non-diffusible substrate that generates a diffusible substance by the enzyme-labeled antibody. (13) The dry immunoassay according to (12), further comprising a reagent layer under the immune reaction layer, the reagent layer containing a low-molecular-weight enzyme that further converts the diffusible substance into a low-molecular product. element. (14) The dry immunoassay element according to (11), further comprising a substrate layer under the immune reaction layer, the substrate layer containing a non-diffusible substrate that generates a diffusible substance by the enzyme-labeled antibody. . (15) Under the substrate layer, the dry immunoassay element according to (14), further comprising a reagent layer containing a low-molecular-weight enzyme that makes the diffusible substance a low-molecular product. . (16) the non-diffusible substrate is a high-molecular-weight polysaccharide, and the enzyme of the enzyme-labeled antibody is an endo-active saccharide-degrading enzyme;
The low-molecular-weight enzyme is an exo-active saccharolytic enzyme.
The dry immunoassay element according to (13) or (15). (17) The dry immunoassay element according to (16), wherein the low-molecular product is glucose. (18) The reagent composition in which the low-molecular product reacts with the low-molecular product to generate a dye having visible absorption is contained in the reagent layer or another water-permeable layer (13) or ( The dry immunoassay element according to 15). (19) The dry immunoassay element according to (11) to (18), wherein the monoclonal antibody is a Fab ′ fragment. (20) The dry immunoassay element according to (11) to (18), wherein the monoclonal antibody is an F (ab ') ₂ fragment. (21) The dry immunoassay element according to (11) to (18), wherein the monoclonal antibody is a Fab fragment.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of one embodiment of an immunoassay element of the present invention.

FIG. 2 is a block diagram of another embodiment of the immunoassay element of the present invention.

FIG. 3 is a calibration curve showing the results of a homogeneous enzyme immunoassay (wet method) performed using the enzyme-labeled antibodies of Example 1 and Comparative Examples 1 and 2.

FIG. 4 is a diagram showing a calibration curve of an immunoassay element of Example 2. The antigen-antibody reaction is performed in a solution system, and only the enzyme reaction is performed in the element.

FIG. 5 is a diagram showing a calibration curve of an immunoassay element of Example 3. Both the immune reaction and the enzyme reaction are performed within the element.

[Explanation of symbols]

 Reference Signs List 10 translucent support 12 reagent layer (or detection layer) 14, 14A immunoreactive layer 16 substrate layer

Claims (3)

(57) [Claims] In a homogeneous enzyme immunoassay method for measuring the amount of an antigen by measuring a change in enzyme activity caused by a reaction between an antigen and an enzyme-labeled antibody, two or more antigen-recognizing methods for recognizing different epitopes of the antigen are provided. A homogeneous enzyme immunoassay method using an enzyme-labeled antibody having a monoclonal antibody and an enzyme. 2. An enzyme-labeled antibody for a homogeneous enzyme immunoassay, comprising a conjugate of two or more monoclonal antibodies recognizing different epitopes of an antigen with an enzyme. 3. A dry immunoassay element for measuring an amount of an antigen by measuring a change in enzyme activity caused by a reaction between the antigen and an enzyme-labeled antibody, wherein two or more monoclonal antibodies recognizing different epitopes of the antigen. A dry immunoassay element comprising an immunoreactive layer having an enzyme-labeled antibody having an enzyme and an enzyme.